The hallmark of cellular aging is the failure of senescent cells to enter/complete the S phase of the cell cycle. The cause for such failure may hold the key for our outstanding the molecular basis of cellular aging. Our previous results demonstrated no difference in the levels of c-myc mRNA and ornithine decarboxylase (ODC) mRNA, but a marked reduction of thymidine kinase (TK) gene expression in old IMR_90 cells as compared to that of the young cells. In addition to TK, all other G1/S genes examined, including thymidine synthase, dihydrofolate reductase, PCNA, histones 1, 3, and 4, exhibited marked attenuation of gene expression in senescent cells, suggesting that such an attenuation may represent a global change critically associated with cellular aging. It is likely that the control of these G1/S genes may play a fundamental role in aging. Preliminary studies using transient expression assay suggest that the age-dependent attenuation of TK gene expression is transcriptionally regulated. Gel mobility shift assay using a 67-bp fragment containing an inverted CCAAT box in the TK promoter suggested that a nuclear trans-acting factor named as CBP-II (CCAAT-Binding Protein II) may be involved in this regulation. CBP-II binding is serum- inducible and age-dependent, prominent in young but not in old IMR-90 cells. CBP-II also appears to be labile, with a half-lifeless than 30 minutes. The main objective of this proposal is to study the role of CBP-II, a newly found trans-acting factor, in the regulation of TK gene. To achieve this goal, we plan to characterize and purify CBP-II using sequence-specific DNA affinity chromatography and gel mobility shift assay. We will also use various screening procedures, including the use of recognition site probes (e.g. the 67-bp) to obtain the CBP-II cDNA. We will use both sense and antisense CBP-II cDNA to construct recombinant plasmids with appropriate DNA expression vectors. Stably transfected IMR_90 cells using these plasmids will allow us to evaluate the role of CBP-II in regulating TK gene expression. In addition, these constructs may also enable us to investigate whether CBP-II is involved in regulating other G1/S genes. Trans-acting factor CBP-II may be critically involved in the regulation of TK gene during aging. The proposed study should yield a deeper understanding of the mechanism of TK gene regulation. It is possible that trans-acting factors such as CBP-II may play a key role in regulating G1/S genes during cellular aging.